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LECTURE 4:. Cloning & PCR. Biotechnology; 3 Credit hours Atta- ur - Rahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST). Polymerase Chain Reaction. Polymerase : DNA polymerase DNA polymerase duplicates DNA - PowerPoint PPT Presentation
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Cloning & PCR
LECTURE 4:
Biotechnology; 3 Credit hours
Atta-ur-Rahman School of Applied Biosciences (ASAB)National University of Sciences and Technology (NUST)
Polymerase Chain Reaction
• Polymerase: DNA polymerase– DNA polymerase duplicates DNA– Before a cell divides, its DNA must be
duplicated• Chain Reaction: The product of a reaction
is used to amplify the same reaction – Results in rapid increase in the product
Polymerase Chain Reaction (PCR)
• PCR performs the chemistry of DNA duplication in vitro
• Numerous PCR applications make this process a staple in most biology laboratories
• Understanding properties of DNA polymerases helps understanding PCR
Discovery• PCR was discovered by Kary Mullis– On a long motorcycle drive– According to Mullis, he was driving his vehicle late night with his
girlfriend– Mentally visualized the process
• Nobel Prize in Chemistry– 1993
DNA polymerase
• Duplicates DNA• Necessary for reproduction of new cells• More than one DNA polymerases exist in
different organisms
Properties of DNA polymearse
3’ 5’
5’ 3’
• Needs a pre-existing DNA to duplicate– Cannot assemble a new strand from
components– Called template DNA
• Can only extend an existing piece of DNA– Called primers
Properties of DNA polymearse
• DNA strands are anti-parallel– One strand goes in 5’ 3’– The complementary strand is opposite
• DNA polymerase always moves in one direction (from 5’ 3’)
3’ 5’
5’ 3’
Properties of DNA polymearse
• DNA polymerase incorporates the four nucleotides (A, T, G, C) to the growing chain
• dNTP follow standard base pairing rule
3’ 5’
5’ 3’
dCTP
dTTP
dCTPdGTPdATP
dGTP
dCTP
dTTP
dATP
dGTP
dCTP
dTTP
dATP
dATP
dGTPdCTP dTTPdATP dGTP
dATP
dGTP
dTTP
dATP
dCTP
dTTP
Properties of DNA polymearse
• The newly generated DNA strands serve as template DNA for the next cycle
• PCR is very sensitive• Widely used
Setting up a PCR Reaction
• Add template DNA and primers• Add dNTPs• Add DNA polymerase
3’ 5’
5’ 3’
dCTP
dTTP
dCTPdGTPdATP
dGTP
dCTP
dTTP
dATP
dGTP
dCTP
dTTP
dATP
dATP
dGTPdCTP dTTPdATP dGTP
dATP
dGTP
dTTP
dATP
dCTP
dTTP
Properties of DNA polymearse
• DNA polymerase needs Mg++ as cofactor• Each DNA polymerase works best under
optimal temperature, pH and salt concentration
• PCR buffer provides optimal pH and salt condition
Taq DNA polymerase
• Derived from Thermus aquaticus• Heat stable DNA polymerase• Ideal temperature 72C
Thermal Cycling
• A PCR machine controls temperature• Typical PCR go through three steps–Denaturation–Annealing– Extension
Denaturation
• Heating separates the double stranded DNA– Denaturation
• Slow cooling anneals the two strands– Renaturation
Heat Cool
Annealing
• Two primers are supplied in molar excess
• They bind to the complementary region
• As the DNA cools, they wedge between two template strands
• Optimal temperature varies based on primer length etc.
• Typical temperature from 40 to 60 C
Extension
• DNA polymerase duplicates DNA• Optimal temperature 72OC
PCR Amplification
Exponential Amplification of template DNA
Typical PCR mix
In a thin wall Eppendorf tube assemble the following
PCR components Amount
Template DNA (5-200 ng)1 mM dNTPs (200 uM final)10 X PCR buffer25 mM MgCl2 (1.5 mM final)20 uM forward primer (20 pmoles final)20 uM reverse primer (20 pmoles final)5 units/uL Taq DNA polymerase (1.5 units)WaterFinal Volume
variable10 uL5 uL3 uL1 uL1 uL0.3 uLVariable50 uL
Applications
• Revolutionized how we study biology– Recombinant DNA Technology– Research– Diagnostics– Forensics